CN105308173A - A pharmaceutical composition for stimulation of angiogenesis - Google Patents

Abstract

Provided is a pharmaceutical composition for growth induction in blood vessel tissue which contains purified plasmid DNA encoding a vascular endothelial growth factor (VEGF) and pharmaceutically acceptable excipients that include a cryoprotectant as a vehicle and/or a pH stabilizer for stabilizing the pH in the range of 7.0 to 9.0, in effective quantities. Also provided is a storage method of plasmid DNA which encodes a VEGF comprising mixing the purified plasmid DNA with a solution of at least one cryoprotectant having properties of a vehicle and/or a pH stabilizer in the pH range of 7.0-9.0. The solution is lyophilized and stored at +2 DEG C to +8 DEG C. Supercoiled DNA pCMV-VEGF165 may be used which is produced by culturing Esherichia coli strain TOP10/pCMV-VEGF165. The pharmaceutical composition is administered to a human in quantities sufficient to provide a necessary therapeutic effect. The provided pharmaceutical composition of plasmid DNA pCMV-VEGF165 does not change significantly properties of the active substance when stored for a long time at a temperature from +2 DEG C to +8 DEG C.

Description

For stimulating the pharmaceutical composition of vasculogenesis

Technical field

Object of the present invention belongs to biotechnology, the i.e. strain producer of plasmid DNA, one can at the relevant pharmaceutical composition of injection site inducing angiogenesis (vascularization), a kind of and the application in the treatment of the composite treatment at atherosclerotic's lower limb ischemia and the wound at a variety of causes and ulcer, and method storing the plasmid DNA of purifying.

Background technology

Adopt naked plasmid dna be used for the treatment of disease or for pathogenic agent or tumour cell antigen and vaccinated gene therapy makes have developed in adverse conditions, especially can be stored at the temperature of positive temperature or increase, transport and the final formulation of the therapeutic DNA used by expert.The physics and chemistry stability of plasmid DNA stored is subject to the composition of vehicle, its concentration under final formulation and/or condition of storage and/or the remarkable restriction (SchleefM. of content, SchmidtT., studies and clinical application (Animal-freeproductionofccc-supercoiledplasmidsforresearc handclinicalapplications) is used for without animal productiong ccc-super spirial plasmid, J.GeneMed., 6Suppl1:S45-53 (2004)).The main process of the pharmaceutical active form of the plasmid DNA that impact stores is the degraded of DNA chain, and it causes forming lax cyclic plasmid DNA and forming linear double chain form subsequently.It is known to freezing plasmid DNA solution is stored at the temperature lower than-80 DEG C, the degraded of super coiled DNA form is almost (WaltherW. inconspicuous, SteinU., VossC., SchmidtT., SchleefM., SchlagP.M., the stability analysis of naked DNA standing storage: for the impact (Stabilityanalysisforlong-termstorageofnakedDNA:impactonn onviralinvivogenetransfer) of non-viral vivo gene transfer, AnalBiochem., 318 (2): 230-5 (2003)).The method of this storage plasmid DNA cannot widely use in clinical manipulation, because in most of the cases, medical institutions and pharmacy unrequired refrigerating plant.Transport final formulation and to maintain the low temperature like this of this product very difficult.Select suitable vehicle composition and concentration that plasmid DNA solution may be made to stablize at being stored in 4 DEG C maybe to stablize more than 3 years (PrzybylowskiM. at being stored in-20 DEG C for 12 months, BartidoS., Borquez-OjedaO., SadelainM., RiviereI., for the production (Productionofclinical-gradeplasmidDNAforhumanPhaseIclinic altrialsandlargeanimalclinicalstudies) of the clinical grade plasmid DNA of mankind I clinical trial phase and macrofauna clinical study, Vaccine, 25 (27): 5013-24 (2007)).

The most popular method developing more stabilizer type is lyophilization.Usually, freeze-dried formulation stablizes the several years at 4 DEG C, and in some cases, can by product stock room temperature several months or and even 2 years.Lyophilization also allows the concentration changing active substance, wherein bottle can be filled a small amount of concentrated solution, volume can be increased to and dissolve necessary level for product.The freeze-drying of hypotonic solution can be used, subsequently by the solubilize (AnchordoquyT.J. of lyophilized products salt solution or normal osmotic pressure, ArmstrongT.K., MolinaM.C., LMD stabilizes the non-virus carrier under Hyposmolality during freeze-drying: the volume (Lowmolecularweightdextransstabilizenonviralvectorsduring lyophilizationatlowosmolalities:concentratingsuspensions byrehydrationtoreducedvolumes) by rehydration, suspension being concentrated into reduction, JPharmSci., 94 (6): 1226-36 (2005)).

Usually, freeze-drying plasmid DNA solution adds the stability that product stores further, but superhelix (AnchordoquyT.J. that is freezing and vacuum-sublimation step possibility grievous injury plasmid DNA, ArmstrongT.K., MolinaM.C., AllisonS.D., ZhangY., PatelM.M., Deng, the physically stable (PhysicalstabilizationofplasmidDNA-basedtherapeuticsdurin gfreezinganddrying) of plasmid DNA base therapeutical agent between freezing and dry epoch, at " CostantinoHR, PikalM.J. edit, biologics freeze-drying (Lyophilisationofbiopharmaceuticals), AAPS press " in, pp.605-41 (2004)).Particularly, the freezing aqueous solution never containing vehicle carries out the elimination that DNA freeze-drying causes the hydration coating of coordinated water molecule and DNA molecular, the structural integrity of DNA molecular is caused to lose (PoxonS.W., HughesJ.A., lyophilization is on the impact (TheeffectoflyophilizationonplasmidDNAactivity) of plasmid DNA activity, PharmDevTechnol., 5 (1): 115-22 (2005)).The result comprising the DNA molecular configuration of the degraded of complementary key between nitrogenous base and the Partial digestion of accumulation impaired causes undesirable event, namely, the biological activity of plasmid DNA reduces up to 25% (AnchordoquyT.J. from original activity, ArmstrongT.K., MolinaM.C., LMD stabilizes the non-virus carrier under Hyposmolality during freeze-drying: the volume (Lowmolecularweightdextransstabilizenonviralvectorsduring lyophilizationatlowosmolalities:concentratingsuspensions byrehydrationtoreducedvolumes) by rehydration, suspension being concentrated into reduction, JPharmSci., 94 (6): 1226-36 (2005)).The loss of the known coordinated water molecule by distillation removing offsets (MaitaniY. by adding non-volatile hydrophilic substance such as sugar and polyvalent alcohol to Solutions in Freeze-drying, AsoY., YamadaA., YoshiokaS,. carbohydrate is for the effect (Effectofsugarsonstoragestabilityoflyophilizedliposome/DN Acomplexeswithhightransfectionefficiency) of stability in storage of freeze-dried lipidosome/DNA mixture with high transfection efficiency, IntJPharm., 356 (l-2): 69-75 (2008)).To stablize in field liposome (the lyposome) (United States Patent (USP) 7 that the known method of major part uses the freeze-dried containing DNA being carried out DNA by lyophilization, 323,297), and therefore, currently known methods is doubtful for the suitability of the solution containing naked plasmid dna.At QuaakS., HaanenJ., BeijnenJ., andNuijenB., naked plasmid dna preparation: different disaccharides is for the impact (NakedPlasmidDNAFormulation:EffectofDifferentDisaccharide sonStabilityafterLyophilisation) of freeze-drying rear stability, AAPSPharmSciTech, Vol.11, in the research of No.1 (in March, 2010), have detected the impact of multiple polysaccharide for the freeze-drying of plasmid DNA, its specify that sucrose and DNA in mass ratio 20:1 use the formulation provided for the most stable storing.This research does not consider that pH and salt solution are for the impact of plasmid DNA stability, and do not study isotonic DNA solution in concentration lower than composition during 5mg/ml, also not studying may potentially for obtaining the monose of stable freeze-dried macromole prepared product.

Summary of the invention

Object of the present invention belongs to pharmaceutical composition feasible on the strain producer of plasmid DNA and physiology, and it provides the stability of the final formulation of plasmid DNA for a long time and can be used for gene therapy.

Strain producer's bacillus coli (Esherichiacoli) of plasmid DNA is TOP10/pCMV-VEGF165 bacterial strain, it comprises plasmid pCMV-VEGF165, and the super coiled DNA of plasmid pCMV-VEGF165 is produced when cultivating in the medium, wherein this bacterial strain has been deposited in Russia agricultural Organism Depositary (RussianCollectionofAgriculturalMicroorganisms) RCAMARRIAMRAAS of complete Russian agricultural microbiology institute (All-RussiaResearchInstituteforAgriculturalMicrobiology), preserving number is 517.

Pharmaceutical composition is included in human cell the prepared product of the plasmid DNA of the purifying of encoding VEGF (VEGF) under the control of the functional genetic element providing genetic expression, and for the pharmaceutically acceptable vehicle of at least one of the significant quantity providing isotonic solution, the pharmaceutically acceptable vehicle of wherein said at least one is at least one cryoprotector, described cryoprotector is vehicle (vehicle), pH stablizer or its combination, the concentration of the plasmid DNA of wherein said purifying is from 0.1 to 10mg/ml, and the pH of composition is 7.0 to 9.0.Described composition is used in induced growth in vascular tissue.

Another object of the present invention is the method for the plasmid DNA of the purifying storing encoding VEGF (VEGF), the described method plasmid DNA comprised to purifying adds the solution of at least one cryoprotector, thus obtain isotonic solution, subsequently by isotonic solution freeze-drying, and lyophilized products is stored in from the temperature of+2 DEG C to+8 DEG C, wherein before freeze-drying, the concentration of plasmid DNA purification is 0.1 to 10mg/ml, and the pH of isotonic solution is 7.0 to 9.0.

Described pharmaceutical composition can be used for the injection solution for the preparation of intramuscular, intravenously or intra-arterial, subcutaneous and intradermal administration.It can be used as gel and is placed on absorbability or nonabsorable carrier for percutaneous drug delivery.

Described pharmaceutical composition be intended to for interim (extempore) prepare solution or not then for experimenter's (patient, the wounded, animal) intramuscular or any other endogenous administration with induction of vascular tissue development.

A therapeutic indication of described pharmaceutical composition is the ischemic condition of the various cause of disease, comprises coronary heart disease, lower limb chmnic. obstructive artery disease; Need prosthetic organizational process to carry out the situation of correcting, such as comparatively large, persistence wound (ulcer), comprises burn, local damage, comprises fracture and bone defect.

Another object of the present invention uses the method for described pharmaceutical composition, and described method comprises uses the described pharmaceutical composition of significant quantity to provide the therapeutic action depending on classification of diseases form (nosologicform) and medical indications to human experimenter.

Accompanying drawing explanation

By easily obtaining, embodiment and the advantage that much accompanies thereof are understood more fully, because to combine when accompanying drawing is considered this and will become by reference to following detailed description and be easier to understand, wherein:

Fig. 1 shows the expression map of plasmid pCMV-VEGF165 (length is 4859 base pairs).Employ following abbreviation: the promotor/enhanser region of the early gene of " CMV early promoter/enhanser region "-cytomegalovirus; Enhanser region; The promoter region of " CMV promoter "-cytomegalovirus; " TATA box "-TATA-element; The point of " transcription initiation "-transcription initiation; " Kozak "-Kozak sequence; First codon of the open reading frame of " initiator codon "-VEGF165; The polypeptide open reading frame (ORF) of " VEGF165 "-rhVEGF; " terminator codon "; " SV40pA item "-polyadenylation signal and virus terminator SV40; The annealing region of " CMV forward primer "-standard primer CMV forward; The annealing region of " M13 forward 20 (forward20) primer "-standard primer M13 forward 20; The annealing region of " M13pUC forward primer "-standard primer M13pUC forward; The starting area of " pBR322 starting point "-plasmid replication pBR322; The starting area of " fl starting point "-phage replication fl; The prokaryotic promoter of " AmpR promotor "-gene bla; " NTPII (neomycin phosphotransferase; " KanR "-coding provides the sequence of the aminoglycoside-3'-phosphotransferase of the bacterial resistance to kantlex.Arrow shows the direction of genetic transcription, provides the numbering of first and last nucleotide fragments in bracket.The recognition site of restriction enzyme provides with italic, gives the Nucleotide in cleavage site in bracket.

Fig. 2 shows the angiogram of the 74 years old patient suffering from Limb chronic ischemia.Diagnosis: atherosclerosis, bilateral stock ilium occlusive disease stage IIb-III (during rest pain).Value when being admitted to hospital: AAi-0.48 (right side), 0.32 (left side); Transcutaneous oxygen pressure-61mmHg.This patient accepted the part of physiatrics as standard summary treatment (dextran, disaggregant).Value when 90 days: walking distance-130m; AAi-0.5 (right side), 0.57 (left side); Transcutaneous oxygen pressure-78mmHg.Angiogram before A, B-treatment: femur top and middle 1/3rd places (A); Shin bone (B); C, D-administration Neovasculgen is as the angiogram after a part for composite treatment 90 days time: femur top and middle 1/3rd places (C); Shin bone (D).Show the gratifying blood vessel filled up with arrow, comprise the collatoral vessel in formation.

Embodiment

All publications mentioned in this article, patent application, patent and other reference with its full content by reference to being incorporated to herein.In addition, unless specifically stated so, otherwise material, method and embodiment are only exemplary, and do not plan to be a kind of restriction.The plasmid DNA of encoding VEGF is by being connected the open reading frame (ORF) of mankind cDNAVEGR with plasmid vector and plasmid replication and obtaining in bacillus coli cell, and described plasmid vector provides the expression of coded gene in human cell.The example of carrier can be plasmid pcDNA, pCMV-Script and pBK-CMV (source-HTTP: the electronic databank at //addgene.org/vector-database/ place).The necessary assembly of carrier is eukaryotic cell promotor.The limiting examples of promotor includes but not limited to cytomegalovirus early promoter/enhanser (CMV), human translation EF-1 α (EF1a) promotor, human ubiquitine C (Ubc) promotor, simian virus 40 (SV40) promotor, mouse source phosphoglyceric kinase 1 (PGK) promotor and human B-actin promoter.Preferred group of carrier for VEGF expression is the carrier being suitable for human cell, its region of replication orgin comprising the immediate early promoter (hereinafter-CMV promoter) of cytomegalovirus, antibiotic resistance genes and provide medium or high plasmid copy number, it can be the replication orgin of such as CoIEl, pUC, pBR322 or pl5A.More preferably the group of carrier comprises encoding neomycin phosphotransferase (NPTII), provide gene to microbiotic kalamycin resistance, and this kind of gene makes it possible to from the process manufacturing plasmid, get rid of the microbiotic using Ampicillin Trihydrate group.As the carrier for obtaining target plasmid DNA, the plasmid of the ORF of other genes of coding can be used.Can by using the ORF region of restriction enzyme removing heterologous gene, be separated receptor DNA fragment and subsequently its donor DNA segment with the ORF of the mankind cDNAVEGF of encoding be connected, thus obtain target plasmid DNA from these plasmid DNA.An example of receptor plasmid can be plasmid pEGFP-N2.The region of the open reading frame of mankind cDNAVEGF can be selected from such as coding and have the known splice variant of four of the VEGF isotype of 121,145,165 or 189 amino acid lengths.The splice variant with 165 amino acid lengths for obtaining the preferred VEGF isotype of plasmid DNA.The ORF region of mankind cDNAVEGF may derive from total cDNA of human tissue or (overlaying) oligonucleotide synthesis from superposition.

In the composite part of the ORF of mankind cDNAVEGF, a part of or all codons can be replaced by degenerate codon.If the fragment of the synthesis of cDNA provides the efficiency that lifetime of the cDNA comparable with native gene and target gene are translated, then replaceable natural areas.CDNA for obtaining the VEGF of target plasmid can comprise known polymorphism (replacement of the Nucleotide of separation), and it does not change the amino acid composition of coded polypeptide.The use of the cDNA of the VEGF containing this polymorphism does not change the character of derived target plasmid.Coding VEGF is also intended to be the connection product of the ORF in the VEGF region with 165 amino acid whose isotypes and the receptor fragments of plasmid pBK-CMV for the preferred plasmid of medical usage.

The plasmid DNA pCMV-VEGF165 length described in patent RU2297848 is 4859 base pairs, and nucleotides sequence is classified as SEQIDNO:1.The structure of plasmid pCMV-VEGF165 provides in FIG.

The plasmid with 4859 base pairs comprises:

1. the region between Nucleotide 1-361, from carrier pBK-CMV Nucleotide 968-4859 between sequence, it comprises:

1.1. provide the element of the expression of target gene in mammalian cell: CMV early promoter/enhanser region (Nucleotide 4626-355), it comprises enhanser region (Nucleotide 4684-231) and cytomegalovirus promoter (Nucleotide 274-355); TATA box-element (Nucleotide 320-326); Transcripting start point (349); Polyadenylation signal and viral SV40 terminator (Nucleotide 1306-1531); And

1.2. maintain the element that plasmid exists in bacterial cell, start the region fl (Nucleotide 1583-1995) of phage replication; The prokaryotic promoter (Nucleotide 2058-2086) of gene bla; Coding provides the sequence (Nucleotide 2519-3313) of the aminoglycoside-phosphotransferase of the bacterial resistance to kantlex; And the region of the plasmid replication of pBR322 (Nucleotide 3907-4526);

2. the region between Nucleotide 362-967, it comprise be positioned at target gene initiator codon near and the Kozak sequence (Nucleotide 380-391) of the translation initiation of the mRNA of target gene is provided, the open reading frame (Nucleotide 392-964) of the gene of coding VEGF165, and terminator codon (Nucleotide 965-967).

Plasmid contains restriction endonuclease NdeI (1); BamHI (364); EcoRI (373); BsrGI (854); XmaI (969); Acc651 (978); BelI (1307); NarI (2650); ApaLI (4254); With the recognition site of PciI (4568).

Plasmid (SambrookJ is obtained by using the cloning region of engineered currently known methods, commercially available plasmid and mankind cDNA, FritschEF, ManiatisT. molecular cloning (MolecularCloning). second edition .NewYork, NY: CSH Press (ColdSpringHarborLaboratoryPress); 1989).

The position that plasmid component is placed according to them is listed.The mutual alignment of functional element is very important for effective plasmid activity.

As recombinant plasmid, according to object of the present invention, the various plasmids of the gene containing the VEGF165 that encodes under eukaryotic promoter control can be used.

The DNA fragmentation of identical controlling element of encoding obtains by the nucleotide sequence of modifying DNA fragment (SEQIDNO:1), such as use site-directed mutagenesis modify, the one or more Nucleotide wherein in some site can deleted, insert or increase.As mentioned above, the DNA fragmentation of modification obtains by using the known treatment method generating sudden change.

Producing bacterial strain for obtaining, plasmid DNA pCMV-VEGF165 can be inserted (conversion) and entering in bacterial cell, preferably insert in the escherich's bacillus species (Escherichiasp.) to the transforming susceptible by this kind of plasmid.The selection of the cell be converted is not crucial, because the method transformed and mode are all known.Although depend on cell type and the culture condition of the transformant of acquisition, in bacterial suspension, the abundance of plasmid pCMV-VEGF165 and total content can be different, if recipient cell successful conversion, then will there will be target plasmid.

Plasmid cell transforms and represents that use currently known methods is by plasmid transfered cell.Method for transformation comprises and is such as described in JacA.Nickoloff, microorganism electroporation (molecular biology method) (ElectroporationProtocolsforMicroorganisms (MethodsinMolecularBiology)) // Humana press (HumanaPress); Method in 1st edition (August 15 nineteen ninety-five).

According to an embodiment, " producer of bacterial cell-plasmid CMV-VEGF165 " comprises the bacterial cell having the ability to maintain, copy and gather plasmid CMV-VEGF165 when being cultivated in the medium by bacterial cell.Term " producer of bacterial cell-plasmid pCMV-VEGF165 " refers to and can gather plasmid pCMV-VEGF165 to being no less than 1mg/l (or 1 μ g/10 ⁹individual cell), be more preferably no less than the cell of 10mg/l.Plasmid pCMV-VEGF165 is accumulated in cell, is preferably superhelix annular form.

Preferably, Escherichia bacterium can be used for using the plasmid pCMV-VEGF165 of encoding gene VEGFR under the control of a cmv promoter to transform.The not concrete restriction of type of promotor can be myocyte, inoblast and endotheliocyte, such as, in SV40 or EF-1 activated promotor.

Term " Escherichia bacterium " refers to that described bacterium comprises the escherich's bacillus species according to classification known in microbiology.Can mention that bacillus coli (E.coli) is as the microorganism belonging to escherich's bacillus species.

The escherich's bacillus species that can be used for transforming is not restricted, the limiting examples of wherein said bacterium is described in by Neidhardt, F.C. (bacillus coli and Salmonella typhimurium (EscherichiacoliandSalmonellatyphimurium) in the book of people is waited, AAM (AmericanSocietyforMicrobiology), WashingtonD.C., 1208, table 1).

Preferred F-strain for the production of plasmid pCMV-VEGF165 is the Escherichia coli strain derived from non pathogenic strain K12, and described non pathogenic strain K12 comprises the inactivated gene recA1 of DNA repair system and the inactivated gene endAl of endonuclease.The example of these bacterial strains is DH5 α, DH10B, XL-1 indigo plant and TOP10.

Example for the production of the F-strain of plasmid pCMV-VEGF165 includes but not limited to bacillus coli TOP10.Bacterial strain bacillus coli TOP10 is characterized by cultivation form as described below, physiology and chemistry sign and hereditary feature.

Bacterial strain bacillus coli TOP10/pCMV-VEGF165 has been deposited in the Russia agricultural Organism Depositary RCAMARRIAMRAAS (ARRIAM of complete Russian agricultural microbiology institute on February 24th, 2009,3Podbelskychausse, St.Petersburg, Pushkin8,196608, and to authorize preserving number be 517 RU).

The cultivation morphological characteristic of bacillus coli TOP10 is: the Gram-negative bar forming chain; In nutrient agar-form the bacterium colony at transparent, translucent, convex surface, very medium and edge evenly (even-edged).Bacterial strain is stored in the Luria-Bertrani substratum containing 1% glucose and 10% glycerine.Strain culturing is in Luria-Bertani substratum.

The hereditary property of bacterial strain bacillus coli TOP10 is: the genotype-Δ (araA-leu) 7697 of bacterial strain, [araD139] B/r, Δ (codB-lacI) 3, 80dlacZ58 (M15), galK0, mcrA0, galU-, recAl, endAl, nupG-, rpsL-(strR), Δ (mcrC-mrr) 715.

The conversion that bacterial strain bacillus coli TOP10 plasmid pCMV-VEGF165 carries out causes generating produces bacterial strain TOP10/pCMV-VEGF165, when being cultivated in flask to be added with jolting 12-20 in the Luria-Bertrani substratum that 30 μ g/ml kantlex reach constantly little, the plasmid pCMV-VEGF165 of the amount of its biosynthesizing 5-20mg/l, the plasmid pCMV-VEGF165 being wherein no less than 70% exists with supercoiled form.

The method preparing the plasmid pCMV-VEGF165 of High Purity comprises above-mentioned microbial culture in the substratum of the prokaryotic cell prokaryocyte growth being suitable for transforming; Select cellular biomass; Cell described in resuspension; Alkaline lysis is carried out to cell; Plasmid DNA renaturation is optionally made with acid solution; The agglomerate (pellet) of precipitation separation; Pass through ultrafiltration and concentration; External source impurity and RNA is separated by gel-filtration in containing the solution (such as salt solution) of high salt concentration; By affine (close sulphur) chromatographic separation remaining genomic dna, intracellular toxin and related impurities; Final purifying is carried out by anion-exchange chromatography and subsequent concentration; And by the solution desalination of ultrafiltration/diafiltration by the plasmid pCMV-VEGF165 of purifying.

The prepared product of the plasmid pCMV-VEGF165 obtained, it is suitable for producing pharmaceutical composition and final formulation further, characterizes by following properties:

1) ratio (the lax ring of plasmid and linear forms)-5% (ratio of the concentration of hereinafter-active substance) at the most of related impurities.

2) ratio-at the most 1% of the genomic dna of bacillus coli.

3) ratio-at the most 1% of RNA.

4) ratio-at the most 0.1% of total protein.

5) endotoxin content-50EU/1mg active substance at the most.

Be suitable for the solution of the plasmid DNA pCMV-VEGF165 producing pharmaceutical composition and final formulation further by using other currently known methodss preparation of abstraction and purification DNA, such as, be used in the method for thermo-cracking bacterium when there is washing composition, by the method for calcium chloride selective precipitation RNA, adopt the method for gradient elution separation superhelix and lax plasmid form, with be described in such as D.M.F.Prazeres, " plasmid biopharmaceutics: basis, application and production (PlasmidBiopharmaceuticals:Basics, ApplicationsandManufacturing) ", JohnWiley & Sons, Inc., (2011) additive method in ISBN:978-0-470-23292-7.

The final formulation of plasmid DNA pCMV-VEGF165 should be suitable for intramuscular injection and during standing storage, significantly should not change the character of active substance.The possible final formulation of plasmid pCMV-VEGF165 includes but not limited to frozen soln, liquor, the solution of lyophilized products, i.e. freeze-dried, and amorphous thin film.

In one embodiment, pharmaceutical composition is included in human cell the prepared product of the plasmid DNA of the purifying of encoding VEGF (VEGF) under the control of the functional genetic element providing genetic expression, and for the pharmaceutically acceptable vehicle of at least one of the significant quantity providing isotonic solution, the pharmaceutically acceptable vehicle of wherein said at least one is at least one cryoprotector, described cryoprotector is vehicle, pH stablizer or their combination, the concentration of the plasmid DNA of wherein said purifying is from 0.1 to 10mg/ml, the pH of composition is 7.0 to 9.0.

In another embodiment, pharmaceutical composition comprises the plasmid DNA of purifying and the glucose of significant quantity and sodium phosphate, and for the formation of the isotonic solution of injection, wherein the pH of composition is 7.8.

And in a different embodiment, the pH of composition is 7.2 to 8.5, preferably 7.4 to 8.2.

In one embodiment, the sodium phosphate that described composition comprises the plasmid DNA of purifying, the glucose of 200 to 400mM and 3 arrives 30nM.

In a different embodiment, described pharmaceutical composition comprises the plasmid DNA pCMV-VEGF165 of the purifying of 0.8 to 1.2mg/ml, the dextrose of 280 to 320mM; And the sodium phosphate of 8 to 12mM, wherein the pH of composition is 7.4 to 8.2.

The preferred modification of final formulation is liquor or lyophilized products, because under they can be stored in positive temperature, be namely stored in standard pharmaceutical refrigerator, and do not need the plenty of time to prepare injection.

The more preferred modification of one of final formulation is lyophilized products, has blocked the chemical reaction of the DNA chain degradation causing supercoiled plasmid DNA to transform to lax annular form in default of water potentially.And, when storing the liquid dosage form of plasmid DNA, the Long Term Contact of solution and rubber packing material may occur, and wherein rubber seal thing may comprise extractible transition metal ion potentially, and it can form by catalysis the degraded that hydroxyl radical free radical accelerates DNA chain.

Lyophilized products, i.e. preparation that is amorphous or microcrystalline porous material require that it plays cryoprotector, pH stablizer, sequestrant, antioxidant and/or vectorial effect containing vehicle in the solution treating freeze-drying.The minimum possible group of vehicle can comprise at least one cryoprotector, and described cryoprotector has the character of vehicle, pH stablizer or their combination.Can be that at least one is selected from following component: monose and disaccharides as cryoprotector and vectorial vehicle, polyvalent alcohol, and polymkeric substance, be such as selected from sucrose, lactose, trehalose, N.F,USP MANNITOL, sorbyl alcohol, glucose, raffinose, polyvinylpyrrolidone and its combination.PH stablizer can be that at least one is selected from following component: Trisodium Citrate, sodium phosphate, Tris-HCl, Tris-acetate, glycine, methionine(Met), arginine, Histidine and other amino acid.

When studying the various pharmaceutical composition of plasmid DNA pCMV-VEGF165, contriver surprisingly finds, provides in the combination of stability maximum under the condition of storage of the temperature of+2 DEG C to+8 DEG C by the vehicle of the glucose such as under pH7.8 and sodium phosphate.

When the volume of solution is identical before and after freeze-drying, the composed as follows of solution the best of the character of plasmid DNA pCMV-VEGF165 can be kept at freeze-drying process and in further storing:

1. the concentration of plasmid DNA is from 0.1 to 10mg/ml, preferably from 0.5 to 4mg/ml, more preferably from 0.8 to 1.2mg/ml.

2. the concentration of glucose (dextrose) is from 200 to 400mM, preferably from 250 to 350mM, more preferably from 280mM to 320mM.

3. the concentration of sodium phosphate (mixture of tertiary sodium phosphate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC) is from 3 to 30mM, preferably from 5 to 20mM, more preferably from 8 to 12mM.

4. the pH of solution is from 7.0 to 9.0, preferably from 7.2 to 8.5, more preferably from 7.4 to 8.2.

Structure for the preparation of the plasmid producing bacterial strain and pharmaceutical composition provides in FIG.

Another object of the present invention is the method for the plasmid DNA of the purifying storing encoding VEGF (VEGF), the plasmid DNA comprised to purifying adds the solution of at least one cryoprotector, thus obtain isotonic solution, subsequently by isotonic solution freeze-drying, and lyophilized products is stored in from the temperature of+2 DEG C to+8 DEG C, wherein before freeze-drying, the concentration of plasmid DNA purification is 0.1 to 10mg/ml, and the pH of isotonic solution is 7.0 to 9.0.

In one embodiment, at least one cryoprotector is glucose, and in isotonic solution, the mass ratio of the plasmid DNA of glucose and purifying is 1:50.

In a different embodiment, at least one cryoprotector is glucose, and the ratio of plasmid DNA lax in the storage lyophilized products of 3 months is at the most 10%.

In another embodiment again, at least one cryoprotector is sodium phosphate, and wherein the content of sodium phosphate is from 5 to 20mM, and the pH of isotonic solution is from 7.8 to 9.0.

Another object of the present invention uses the method for described pharmaceutical composition, and described method comprises uses the described pharmaceutical composition of significant quantity to provide the therapeutic action depending on classification of diseases form and medical indications to human experimenter.

Generally describing object of the present invention, by further being understood with reference to some specific embodiment, unless otherwise defined, described specific embodiment is only provided as example object in this article, and being not intended to limit.To the present invention: it is appropriate that the possible implementation of preparation plasmid DNA pCMV-VEGF165 provides embodiment.

Embodiment

The preparation of the solution of the plasmid DNA pCMV-VEGF165 of embodiment 1. purifying.

The preparation of inoculum

Regain the bottle of the inoculum of the preservation of the production bacterial strain TOP10/pCMV-VEGF165 from working cardial cell storehouse from working cardial cell storehouse (workingbank), inoculum is cultivated in the liquid nutrient medium of 50ml.

The biosynthesizing of pDNA

Prepare the fermentor tank of 10 liters of flasks, flask containing substratum is carried out sterilizing, aseptic fixed ducts line and taking-up pipeline, the measurement external member (set) of calibration Connecting quantity, fermentor tank is inoculated and cultivates 8 hours, until realize the constant density (quiescent culture growth phase) of dissolved oxygen with the fixed rate of the agitator of 1000 revs/min, stop oxygenation afterwards and cool flask.The transfer of the sample of culture suspension is used for analyzing culture density.

The production of biomass

In the interval flooring supercentrifuge with centrifuge Fixed Angle Rotor, by biomass, namely cell mass is separated with nutrient solution.The supernatant of nutrient solution is transferred in autoclave and carries out sterilizing and neutralization.The biomass obtained to be retained in refrigerator under low temperature in centrifugal bag.Biomass samples transfer is used for content and the integrity of evaluating objects material.

Suspended biological matter, cracking and neutralization

The biomass of thawing are proceeded in cracking container, and is suspended in aaerosol solution with overhead stirrer.The solution of sodium hydroxide and sodium lauryl sulphate is adopted to mix 5 minutes by overhead stirrer and carry out lysis.When mixing, the solution adding potassium acetate neutralizes, and forms cell debris, the genomic dna of bonding histone and the precipitation of protein simultaneously.Change undissolved sylvite and micelle coagulum into due to sodium lauryl sulphate and define precipitation.Meanwhile, the neutralization of solution causes plasmid DNA renaturation.

The preparation of clarification Methionin

The suspension obtained is proceeded in centrifugal bag, sediment separate out in the interval flooring supercentrifuge with centrifuge Fixed Angle Rotor.The supernatant of nutrient solution is transferred in autoclave and carries out sterilizing and neutralization.Be collected in by the settled solution of plasmid DNA in the feed containers of ultrafiltration external member, described solution also comprises the plasmid DNA of related compound-lax and linear forms and such as remaining genomic dna, RNA, protein and the LAL-intracellular toxin of external source impurity.

Ultrafiltration

Be that the tubular fibre cylinder of 500kDa passes through ultrafiltration and concentration under tangential flowing by the solution of plasmid DNA with retaining threshold value.As a part for ultrafiltration, there are the additional 9 times removals of size less than the molecule of 3kDa equally, such as, the removal of the short-movie section of protein, transport RNA, LAL-intracellular toxin and genomic dna.Solution is concentrated high to 9 times.Concentrated plasmid solution is collected in vial, merges by ultra filtration unit gel-filtration solution washing and with the concentrated solution of plasmid DNA.

Gel-filtration

In first process of macropore dextrin sorbent material Sparse6 quick Flow Meter " GELifesciences " enterprising row by the degree of depth chromatogram purification of gel-filtration.Employ the solution of the high salt concentration containing 2.1M ammonium sulfate and 10mMEDTA-Na, this kind of solution makes it possible to be separated them according to bulk of molecule, and retains the endotoxic impurity of RNA and ALL-due to nonspecific hydrophobic interaction by sorbent material.Impure solution and washing soln are collected and also processes at the appropriate time in container.The solution collection of the semipurified plasmid DNA containing 2.1M ammonium sulfate is entered in Glass Containers.

Affinity chromatography

In second process of sorbent material PlasmidSelectXtra " GELifesciences " enterprising row by the degree of depth chromatogram purification of close sulphur (false affine) chromatogram.In order to wash pillar, use containing the solution of 2.0M ammonium sulfate, and be that the semipurified solution of 2.1M is applied to pillar by ammonium sulfate concentrations.After solution applies, all DNA forms and portion of residual DNA are adsorbed on sorbent material, and wherein residual protein and intracellular toxin are not adsorbed.

After the application, be the ammoniumsulphate soln washing pillar of 2.0M by concentration, wherein the relevant components of residual DNA, genomic dna and plasmid DNA are by wash-out.With the activity eluted material HSCI-01 of ammoniumsulphate soln that concentration is 1.7M.Working concentration is that the sodium hydroxide solution of 0.1M regenerates and purifying pillar.The solution of impurity and washing soln are collected and also processes at the appropriate time in container.The solution collection of half plasmid DNA purification of the ammonium sulfate containing 1.7M is entered in Glass Containers, and dilutes with the ratio of 1:2 with water for injection.

Anion-exchange chromatography

In three process of sorbent material SOURCE30Q " GELifesciences " enterprising row by the degree of depth chromatogram purification of anion-exchange chromatography.Active substance is adsorbed on pillar, and with containing the solution rinsing of 0.4M sodium-chlor.When pillar is by after washed, the positively charged ion of active substance is replaced into sodium ion from ammonium ion, and remaining intracellular toxin is by wash-out.The solution of active substance 1M sodium-chlor is carried out wash-out.Impure solution and washing soln are collected and also processes at the appropriate time in container.The solution collection of the plasmid DNA of purifying is entered in Glass Containers.

Ultrafiltration and diafiltration

Having, to retain threshold value be concentrated by the solution of ultrafiltration by the plasmid DNA of purifying in the external member of the hollow-fiber module of 300kDa.When reaching the concentration of 0.25%, external member being switched to diafiltration pattern, thoroughly changing damping fluid with water for injection.Filtrate collection is entered in container and also process at the appropriate time.Filtrate is poured in Glass Containers, measure the concentration of supercoiled plasmid DNA at lower than the temperature of 70 DEG C.

Embodiment 2. is produced lyophilized products variant and is carried out stability study

In order to obtain the variant of pharmaceutical composition, employ concentration be about the plasmid DNA pCMV-VEGF165 of 2.5mg/ml without salts solution, this solution contains the Tris-HCl of residual content, and pH=7.5, NaCl be 1mM at the most.As cryoprotector, add glucose, sucrose and lactose, it meets the requirement being not less than European Pharmacopoeia requirement.As pH stablizer, employ the sodium radio-phosphate,P-32 solution of pH from 7.2 to 9.0.Employ carbohydrate that final concentration is 300mM and final concentration is the sodium phosphate of 10mM.The vehicle of these concentration provides the isotonicity of the solution for endogenous administration.In the pharmaceutical composition obtained, final DNA concentration is 1mg/ml.

In 5-ml vial, carry out freeze-drying, it is equipped with semi-open freeze-drying rubber seal thing and the test soln of 1.2ml is housed.Shown in the lyophilization scheme of all samples table 1 below.

Table 1. lyophilization scheme

Stage	Time length, minute	Temperature, DEG C
			1) solution is freezing:
a)	30	-50
			b)	300	-50
2) freeze-drying
			a)	60	-30
b)	930	-30
			c)	60	-10
d)	480	-10
			e)	60	20
f)	360	20
			3) finally dry
a)	60	40
			b)	600	40

The pressure of work chamber in drying process is 60 microbars, and product temperature when the freezing stage completes should be not more than subzero 45 DEG C, namely lower than the second-order transition temperature (Tg) 2 DEG C of glucose, and lower than the Tg of every other carbohydrate more than 10 DEG C.Product temperature after freeze drying should lower than 15 DEG C.The temperature of product with freezing enter thermocouple measurement in bottle, described bottle has the product simulation thing (simulator) containing all vehicle except DNA.After final drying, battery compression (batterycompression) is adopted to carry out supercharging to bottle under the atmosphere of aseptic dry air.The bottle aluminium cap closure unloaded.

In order to compare the stability of the product when storing at elevated temperatures, employ the sodium radio-phosphate,P-32 solution that pH is 7.8, this solution by by Sodium phosphate dibasic and sodium dihydrogen phosphate with 91.5: 8.5 mol ratio be mixed to get.The closed vial of freeze-drying is stored in the dry thermostat container of+37 DEG C, wherein monthly takes out one group of bottle, lyophilized products is dissolved, measured the ratio of lax plasmid DNA by analysis mode ion-exchange chromatography.Measuring result provides in table 2.

The ratio of the lax plasmid DNA that table 2. stores at elevated temperatures

Result shows, the glucose as cryoprotector provides the minimum degradation rate of supercoiled plasmid DNA under the mass ratio of carbohydrate and DNA is 1:50, pH=7.8.

For the pharmaceutical composition containing glucose, the relation between the stability of supercoiled plasmid DNA and the concentration of the pH of solution and sodium phosphate (pH7.8) is studied.Found that, for from 5 to 20mM the sodium phosphate of various different concns, the stability of supercoiled plasmid DNA does not significantly change (data are not shown).For the various pH of solution, result display (see table 3) for from 7.8 to 9.0 pH scope stability significantly do not change, but the stability of supercoiled plasmid DNA reduces at ph 7.2.

The ratio of the lax plasmid DNA that table 3. stores at the temperature raised and various pH level

pH	0 month	1 month	2 months	3 months
					7.2	2.2％	4.09％	7.63％	15.17％
7.8	2.2％	2.80％	5.30％	10.00％
					8.4	2.3％	2.76％	3.34％	10.69％
9	2.1％	2.51％	2.97％	8.39％

Therefore, according to accelerating storage data, pharmaceutical composition can select the pH scope of composition to be that the concentration of sodium phosphate is from 5 to 20mM from 7.8 to 9.0.The concentration being the ionized phosphate group of DNA of 1mg/ml due to DNA concentration is about 3mM, and the concentration of buffering salt should significantly beyond the total concn of the ionized group of active substance, and therefore the optimum concn of sodium phosphate is chosen as 10mM.The Optimal pH of solution is 7.8, because such value is closest to physiological pH (7.2-7.4).But, should consider that pH should be higher close to 7.2, pH level as far as possible, plasmid stability higher (until 9.0).Perhaps, plasmid DNA saves its stability under storing temp from+2 to+8 DEG C and pH7.0-9.0, and it is enough to be used in gene therapy.

The preparation of the solution of the plasmid DNA of embodiment 3. purifying and the production of final formulation

Plasmid DNA as shown in embodiment 1 obtained before the anion-exchange chromatography stage terminates.Following phases is carried out according to as described below.

Ultrafiltration and diafiltration

Concentrated by the solution of ultrafiltration by the plasmid DNA of purifying in the external member with hollow-fiber module, the threshold value that retains of the film that described hollow-fiber module has is 300kDa.When reaching the concentration of 0.15%, external member being switched to diafiltration pattern, damping fluid thoroughly being become concentration in water for injection is 10mM, pH7.8, sodium radio-phosphate,P-32 solution containing 4.4% (300mM) glucose.Filtrate collection is entered in container and also process at the appropriate time.The solution of concentrated preparation is poured in Glass Containers, measures the concentration of supercoiled plasmid DNA, solution is adjusted to the final DNA concentration of 0.1%.

Sterile filtration

The solution of final material is transferred in category-A clear area, in (depyrogenized) in the source of reducing phlegm and internal heat aseptic 250-ml container, carry out sterile filtration with disc membrane filter, obtain the transfusion meeting GOST19808-86.Select the sample of Quality Control Department to be forwarded to, container rubber seal thing is closed, then uses aluminium cap closure, and the isolated area in warehouse is carried out freezing.

The obtain solution of plasmid DNA solution is thawed, transfers to category-A clear area.Open container, carry out being aseptically filled in the aseptic 250-ml container in the source of reducing phlegm and internal heat the transfusion obtained according to GOST19808-86 with disc membrane filter.Aseptically solution is poured in the aseptic 5-ml Regular Insulin bottle according to GOST19808-86.According to TU38.006.269-90, bottle rubber seal thing is closed, and turn go lyophilize.

Bottle is placed on the support of Freeze Drying Equipment, freezing and to carry out 3-stage vacuum dry at-45 DEG C.According to GOSTP51314-99, place sealer, take out bottle and close with aluminium cap.

The checking of the stability of the final formulation of embodiment 4.pCMV-VEGF165

The bottle of the aseptic freeze-dried thing containing plasmid DNA pCMV-VEGF165 to be stored in the medicine refrigerator of+4 DEG C 2.5 years, to carry out by every 6 months the analysis that primary ions exchange chromatography carries out related impurities ratio.Give analytical results in table 4.

Table 4. maintains the stability of the final formulation at the temperature of+4 DEG C

Therefore, the formulation of plasmid DNA pCMV-VEGF165 is being stablized at least in two years.

The checking of the efficiency of embodiment 5. pharmaceutical composition

The effect of carrying out with the pharmaceutical composition containing Plasmid Constructs and vehicle treating obtains confirmation in the patient suffering from Limb chronic ischemia, is namely unexpectedly better than the treatment with the composition only comprising plasmid (monotherapy) by the result of the medicine composite for curing containing vehicle.In the research relating to 75 patients, determine higher therapeutic efficiency, described patient all suffers from 2a-3 level Limb chronic ischemia (A.V.Pokrovsky, 2004 according to A.V.Pokrovsky-Fontaine; Common recognition (Inter-SocietyConsensusfortheManagementofPeripheralArteri alDisease) (TASCII) .Eur.J.Vase.Endovasc.Surg. between the association of peripheral arterial disease management, oxygen pressure, AAi and linear blood flow rate in posterior tibial artery).

Walking distance. treat after 3 months, the walking distance of the patient of clinical group is 236.49 ± 193.49m (mean distance), within 6 months, is 284.73 ± 242.02m (mean distance) distance of all acquisitions (for from 20 to 1500m).Patient can being increased in for 149.47m in study group of mean distance of no pain walking, and wherein median adds 127.5m, and the difference between value is significant (p=0.006) statistically.

At control group, patient the mean distance of no pain walking can have dropped 1.42m, and median adds 35.00m, and the difference between value is not statistically significantly (p=0.6).Being worth dynamic difference (mean value+150.89, median+92.5) between group is significant (p=0.001) statistically.

Oxygen through skin pressure. in clinical group, there is the trend of stable increase of the mean value of TPO from 76.69 ± 9.96mmHg during first time access to 85.42 ± 10.87mmHg during the 4th access.Control group has contrary dynamic, namely within the identical observation period, has occurred the reduction of mean value from 76.89 ± 55.76mmHg to 75.37 ± 61.57mmHg.Difference between the value observed in clinical group is significant, and the difference in control group is inapparent (p=0.096), and namely as a part for standard care, the value of patient has almost no change.The difference (+8.00) of difference (+10.25) between group of the average T PO value of the increase be recorded to and median is significant (p=0.0001) statistically.Relatively increasing to of class mean: clinical group+12.40 ± 17.69%, control group 2.12 ± 4.38% (p=0.001).

Ankle-arm index. the ABI of target lower limb has the tendency of increase in the patient of clinical group, has the tendency of reduction in the patient of control group.Therefore, in clinical group, baseline value 0.513 ± 0.182 adds 0.057 at treatments period, is 0.57 6 months time.The difference of the value observed between the 4th time and first time access in clinical group is significance (p=0.001).In control group, this index reduced 0.02 unit in 6 months, had namely dropped to 0.438 ± 0.187 from 0.458 ± 0.182.But this change is not (p=0.5) of significance.

Difference between the average A BI value increased between group is+0.077 (median+0.065), and the difference be recorded to is significance.

Linear blood flow rate (the ultrasonic Doppler excusing from death ripple scanning according to posterior tibial artery). in the patient of clinical group, this value has the tendency of increase during studying: in 6 months, mean value adds 8.24 cels, median adds 5.0 cels, namely from 14.95 ± 10.19 cels until 23.19 ± 12.71 cels.

In the patient of control group, described value adds 1.30 cels (from 17.60 ± 6.60 cels when beginning one's study, until 18.90 ± 6.77 cels 6 months time), and median not change, be namely in the level of 20.0 cels.During studying, the difference between group is significant (p=0.005) statistically.

Mean value in clinical group is increased to larger degree (+6.94 cel) compared with the value shown by control group, and median is also (+5.00 cel); Difference in described value is significance (p=0.005) statistically.

Therefore, based on " no pain walking distance " this efficiency standard, 135.3m when this value was accessed from first time in patient is significantly increased to the 248.7m (adding 110.5%) when 6 months, is significantly different from the trend (p=0.001) in control group.

Other efficiency standards:

-ABI-adds 11.11% (p=0.001);

-TPO-adds 11.38% (p=0.001);

-LBFR-adds 55.12% (p=0.001).

Be incorporated to by reference herein at the RU patent application RU2012137126 submitted on August 31st, 21012.

Various improvement and change can be carried out to the present invention according to above-mentioned instruction.Therefore should be appreciated that, in the scope of claims, the present invention can according to except herein specifically describe except alternate manner operate.

Claims (21)

1. bacillus coli (Escherichiacoli) TOP10/pCMV-VEGF165 bacterial strain, it comprises plasmid pCMV-VEGF165, and the super coiled DNA of plasmid pCMV-VEGF165 is produced when cultivating in the medium, wherein said bacterial strain has been deposited in the Russia agricultural Organism Depositary RCAMARRIAMRAAS of complete Russian agricultural microbiology institute, and preserving number is 517.

2., for the pharmaceutical composition of induced growth in vascular tissue, described composition comprises:

The prepared product of the plasmid DNA of the purifying of encoding VEGF (VEGF) under the control of the functional genetic element of genetic expression is provided in human cell, and

The pharmaceutically acceptable vehicle of at least one for the significant quantity providing isotonic solution, the pharmaceutically acceptable vehicle of wherein said at least one is at least one cryoprotector, and described cryoprotector is vehicle, pH stablizer or its combination,

The concentration of the plasmid DNA of wherein said purifying is from 0.1mg/ml to 10mg/ml, and the pH of described composition is 7.0 to 9.0.

3. pharmaceutical composition according to claim 2, the pharmaceutically acceptable vehicle of wherein said at least one is selected from monose and disaccharides, polyvalent alcohol, polymkeric substance and its mixture.

4. pharmaceutical composition according to claim 2, the pharmaceutically acceptable vehicle of wherein said at least one is selected from sucrose, lactose, trehalose, N.F,USP MANNITOL, sorbyl alcohol, glucose, raffinose, polyvinylpyrrolidone and its mixture.

5. pharmaceutical composition according to claim 2, the pharmaceutically acceptable vehicle of wherein said at least one is pH stablizer, and described pH stablizer is that at least one is selected from following component: Trisodium Citrate, sodium phosphate, Tris-HCl, Tris-acetate, glycine, other amino acid and their mixture.

6. pharmaceutical composition according to claim 2, described pharmaceutical composition comprises the plasmid DNA of purifying and the glucose of significant quantity and sodium phosphate, and for the formation of the isotonic solution of injection, the pH of wherein said composition is 7.8.

7. pharmaceutical composition according to claim 2, the pH of wherein said composition is from 7.2 to 8.5.

8. pharmaceutical composition according to claim 2, the pH of wherein said composition is from 7.4 to 8.2.

9. pharmaceutical composition according to claim 2, the concentration of the plasmid DNA of wherein said purifying is from 0.5mg/ml to 4mg/ml.

10. pharmaceutical composition according to claim 2, the concentration of the plasmid DNA of wherein said purifying is from 0.8mg/ml to 1.2mg/ml.

11. pharmaceutical compositions according to claim 2, described pharmaceutical composition comprises the glucose from 200mM to 400mM and the sodium phosphate from 3nM to 30nM.

12. pharmaceutical compositions according to claim 2, the plasmid DNA of wherein said purifying is pCMV-VEGF165.

13. pharmaceutical compositions according to claim 12, wherein said composition comprises:

The plasmid DNA pCMV-VEGF165 of the purifying from 0.8mg/ml to 1.2mg/ml;

Dextrose from 280mM to 320mM; And

Sodium phosphate from 8mM to 12mM,

The pH of wherein said composition is from 7.4 to 8.2.

14. pharmaceutical compositions according to claim 13, the nucleotides sequence of the plasmid DNA pCMV-VEGF165 of wherein said purifying is classified as SEQIDNO:1.

15. pharmaceutical compositions according to claim 2, the plasmid DNA being wherein not less than the described purifying of 70% exists with supercoiled form.

16. pharmaceutical compositions according to claim 2, the prepared product of the plasmid DNA of wherein said purifying comprises

The impurity being selected from lax loop type, linear plasmid form and its mixture of 5% at the most,

The genomic dna of 1% at the most,

The RNA of 1% at the most,

The total protein of 0.1% at the most, and

50EU intracellular toxin/1mg active substance at the most.

The method of the plasmid DNA of the purifying of 17. 1 kinds of storages encoding VEGF (VEGF), described method comprises:

Plasmid DNA to described purifying adds the solution of at least one cryoprotector, thus obtains isotonic solution,

Subsequently by described isotonic solution freeze-drying, and

Lyophilized products is stored in from the temperature of+2 DEG C to+8 DEG C,

Wherein before freeze-drying, the concentration of the plasmid DNA of described purifying is from 0.1mg/ml to 10mg/ml, and the pH of described isotonic solution is from 7.0 to 9.0.

18. methods according to claim 17, wherein said at least one cryoprotector is glucose, and wherein in described isotonic solution the mass ratio of the plasmid DNA of glucose and described purifying be 1:50.

19. methods according to claim 18, wherein said at least one cryoprotector is glucose, and the ratio of wherein lax in the lyophilized products storing 3 months plasmid DNA is at the most 10%.

20. methods according to claim 18, wherein said at least one cryoprotector is sodium phosphate, and the content of sodium phosphate is from 5mM to 20mM, and the pH of described isotonic solution is from 7.8 to 9.0.

21. 1 kinds of methods using pharmaceutical composition according to claim 2, described method comprises uses the described pharmaceutical composition of significant quantity to provide the therapeutic action depending on classification of diseases form and medical indications to human experimenter.